Many growth factors and hormones such as nerve growth factor (NGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and insulin mediate their signals through interactions with cell surface tyrosine kinase receptors. The transduction of extracellular signals across the membrane, initiated by ligand binding, leads to the propagation of multiple signaling events which ultimately control target biochemical pathways within the cell.
The phosphatidylinositol 3-kinases (PI3Ks) represent a ubiquitous family of heterodimeric lipid kinases that are found in association with the cytoplasmic domain of hormone and growth factor receptors and oncogene products. PI3Ks act as downstream effectors of these receptors, are recruited upon receptor stimulation and mediate the activation of second messenger signaling pathways through the production of phosphorylated derivatives of inositol (Fry, Biochim. Biophys. Acta., 1994, 1226:237-268).
The class I PI3Ks are composed of a Src homology-2 domain-containing an 85 kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110), which catalyze the phosphorylation of phosphotidylinositol at the D3 position of the inositol ring (Cantley, Science 296:1655-1657 (2002); Carpenter and Cantley, Curr. Opin. Cell Biol., 8:153-8 (1996)).
PI3Ks plays a central role in a broad range of biological effects including growth factor mediated cell transformation, mitogenesis, protein trafficking, cell survival and proliferation, DNA synthesis, apoptosis, neurite outgrowth and insulin-stimulated glucose transport (reviewed in Fry, Biochim. Biophys. Acta., 1994, 1226, 237-268). Its apparent involvement in so many disparate signaling pathways suggests that it may provide a more general, facilitative, signaling function, such as targeting an active complex, rather than directly controlling these myriad events.
Inhibitors of proteins that are involved in the PI3K signaling have been suggested as therapeutic agents. Examples of said inhibitors include wortmannin, demethoxyviridin, quercetin and LY294002. These inhibitors primarily target the p110 subunit and display toxicity and short half-life which limit their use in clinical trials.
An alternative approach to these inhibitors has been to specifically inhibit the expression of important pathways proteins by RNA interference, such as specific inhibition of p85 expression by siRNA.
The aim of the present invention was to find inhibitors of PI3K signaling. Preferably, said inhibitors would present the following advantages: high stability, a cell penetration/diffusion better than siRNAs, and a half-life better than siRNAs.
The inventors made the surprising observation that peptides having a phosphorylated YX1X2X1 motif as defined here after are capable of reducing tumor size in vivo. Sequences having phosphotyrosine residues in the context of the motif YMXM are known to bind to SH2 domains. p85 is described to possess two SH2 domains and the binding of these SH2 domains to phosphotyrosine residues in the context of the motif YMXM is described to activate p85 and p110 leading to the catalyzation of the phosphorylation of phosphatidylinositol (PdtIns) producing PtdIns(3), PtdIns(3,4)P2 and PtdIns (3,4,5)P3.
Synthetic peptides containing a phosphorylated YMXM motif are known in the art to activate PI3K in vitro (White et al. 1994 The Journal of Biological Chemistry 269 (7):1-4). As activation of PI3K signaling is known to be implicated in cancer development, the person skilled in the art would thus not have been induced to use peptides activating PI3K signaling for treating cancer.